Perioperative myocardial ischemia and infarction represent the major cause of morbidity and mortality for the large number of surgical patients who undergoes a variety of operations such as heart transplantation, coronary artery bypass grafting, and revascularization. Recent studies have demonstrated that cardiomyocyte apoptosis, or programmed cell death, plays an important role in ischemic myocardial injury. Innate immune system such as Toll-like receptor 4 (TLR4) represents the first line of defense against infection. In addition to its pivotal role in host immunity, recent studies have demonstrated that TLR4 is an important functional contributor to tissue inflammation and cell survival in response to non-infectious injury. Our preliminary data suggest that TLR4 signaling plays a critical role in cardioprotection against ischemic injury in the heart and in isolated cardiomyocytes. The goals of this proposal are to define the role of cardiac TLR4 in protecting cardiomvocvtes in models of ischemia-reperfusion injury (IRI) and to identify the downstream mechanisms that mediate these effects. We anticipate that insights gained from the proposed studies will serve as a foundation for the future development of novel therapeutic approaches for the management of ischemic myocardial injury. This proposal is based on the following three hypotheses: 1) that TLR4 activation via its signaling protein IRAK-1 represents an important survival mechanism in the heart, 2) that nitric oxide synthase 2 (NOS2) mediates the TLR4-induced survival benefits, and 3) that augmentation of cardiac TLR4 signaling will reduce myocardial damage and produce a meaningful functional rescue in IRI. To test these hypotheses, we will manipulate cardiac expression of TLR4 and IRAK-1 using genetically modified animals as well as adenoviral gene transfer. In Specific Aim 1: we will explore the mechanisms by which TLR4 protects cardiomyocytes in in vitro models of apoptosis. In Specific Aim 2: we will determine how NOS2 and NO contribute to the TLR4-mediated anti-apoptotic effects in isolated cardiomyocytes. In Specific Aim 3: we will ascertain the role of cardiac (vs. extra-cardiac) TLR4 in protecting the heart against IRI. In Specific Aim 4: we will evaluate the anatomic and functional consequences of manipulating IRAK-1 in mouse models of IRI. We will test the impact of IRAK-1 deletion or cardiac IRAK-1 expression (via gene transfer) on IRI. Defining the signaling pathways that control cardiomyocyte survival and learning to manipulate these pathways in the heart may provide novel approach for the treatment of some cardiac conditions such as ischemia injury and cardiomyopathy.